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The existence of mGluR, NMDAR, AMPAR and putative KAR heteroreceptor complexes in synaptic and extrasynaptic regions of brain glutamate synapses represents a major integrative mechanism. Our aim in the current article is to analyze if the formation of the different types glutamate hetereceptor complexes involves the contribution of triplet amino acid homologies (protriplets) in a postulated receptor interface based on the triplet puzzle theory. Seven main sets (lists) of receptor pairs in databases were used containing various sets (lists) of human receptor heteromers and nonheteromers obtained from the available scientific publications including the publically available GPCR-hetnet database. Brain mGluR1-mGluR5 and mGluR2-mGluR4 isoreceptor complexes were demonstrated with a predominant extrasynaptic localization at a post- and prejunctional localization. The existence of putative mGluR4-mGluR7 heteroreceptor complexes in the basal ganglia is proposed. Metabotropic glutamate receptor subtypes also participated in the formation of a large number of heteroreceptor complexes like mGluR1-A1R, mGluR5-A2AR, mGluR5-D2R and D2R-A2AR-mGluR5, located in relation to glutamate synapses, especially in the basal ganglia. A putative mGluR1-GABAB1/2 heterocomplex may also exist. NMDAR heteroreceptor complexes were also demonstrated as a fundamental integrative mechanism in the glutamate synapse and its extrasynaptic membranes. It represented fundamental work on inter alia NMDAR-mGluR5, NMDAR-D1R and NMDAR-D2R heteroreceptor complexes involving both antagonistic and facilitatory allosteric receptor-receptor interactions. As to AMPA receptors, a heterocomplex was found for the interaction between IFNgR1 and the AMPAR mediated via the subunit GluA1 which may be of relevance for neuroinflammation. AMPAR-D2R heteroreceptor complexes were also demonstrated. Besides glutamate heteroreceptor complexes and their allosteric receptor-receptor interactions, a significant mechanism for the functional crosstalk can also be phosphorylation and/or reorganization of adapter proteins with dynamic binding to the two receptors modulating the allosteric receptor mechanism.
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Encéfalo/metabolismo , Complejos Multiproteicos/metabolismo , Receptores de Glutamato/metabolismo , Animales , Humanos , Isoformas de Proteínas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Studies on serotonin-selective reuptake inhibitors have established that disturbances in the ascending 5-HT neuron systems and their 5-HT receptor subtypes and collateral networks to the forebrain contribute to the etiology of major depression and are targets for treatment. The therapeutic action of serotonin-selective reuptake inhibitors is of proven effectiveness, but the mechanisms underlying their effect are still unclear. There are many 5-HT subtypes involved; some need to be blocked (e.g., 5-HT2A, 5-HT3, and 5-HT7), whereas others need to be activated (e.g., postjunctional 5-HT1A and 5-HT4). These state-of-the-art developments are in line with the hypothesis that the development of major depression can involve an imbalance of the activity between different types of 5-HT isoreceptors. In the current study, using in situ proximity ligation assay (PLA), we report evidence for the existence of brain 5-HT1A-5-HT2A isoreceptor complexes validated in cellular models with bioluminescence resonance energy transfer (BRET2) assay. A high density of PLA-positive clusters visualizing 5-HT1A-5-HT2A isoreceptor complexes was demonstrated in the pyramidal cell layer of the CA1-CA3 regions of the dorsal hippocampus. A marked reduction in the density of PLA-positive clusters was observed in the CA1 and CA2 regions 24 h after a forced swim test session, indicating the dynamics of this 5-HT isoreceptor complex. Using a bioinformatic approach, previous work indicates that receptors forming heterodimers demonstrate triplet amino acid homologies. The receptor interface of the 5-HT1A-5-HT2A isoreceptor dimer was shown to contain the LLG and QNA protriplets in the transmembrane and intracellular domain, respectively. The 5-HT2A agonist TCB2 markedly reduced the affinity of the 5-HT1A agonist ipsapirone for the 5-HT1A agonist binding sites in the frontal lobe using the 5-HT1A radioligand binding assay. This action was blocked by the 5-HT2A antagonist ketanserin. It is proposed that the demonstrated 5-HT1A-5-HT2A isoreceptor complexes may play a role in depression through integration of 5-HT recognition, signaling and trafficking in the plasma membrane in two major 5-HT receptor subtypes known to be involved in depression. Antagonistic allosteric receptor-receptor interactions appear to be involved in this integrative process.
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The mild neuroinflammation hypothesis of schizophrenia was introduced by Bechter in 2001. It has been hypothesized that a hypofunction of glutamatergic signaling via N-methyl-D-aspartate receptors (NMDARs) and hyperactivation of dopamine D2 receptors play a role in schizophrenia. The triplet puzzle theory states that sets of triplet amino acid homologies guide two different receptors toward each other and contributes to the formation of a receptor heteromer. It is, therefore, proposed that putative NMDAR-C-C chemokine receptor type 2 (CCR2), NMDAR-C-X-C chemokine receptor type 4 (CXCR4), and NMDAR- interleukin 1 receptor type II (IL1R2) heteromers can be formed in the neuronal networks in mild neuroinflammation due to demonstration of Gly-Leu-Leu (GLL), Val-Ser-Thr (VST), and/or Ser-Val-Ser (SVS) amino acid homologies between these receptor protomers. This molecular process may underlie the ability to produce symptoms of schizophrenia in mild neuroinflammation. In this state, volume transmission (VT) is increased involving increased extracellular vesicle-mediated VT from microglia and astroglia. These vesicles may contain CCR2, CXCR4, and/or IL1R2 as well as their ligands and upon internalization by endocytic pathways into neurons can form heteroreceptor complexes with NMDAR in the plasma membrane with pathological allosteric receptor-receptor interactions involving increased internalization and reduced NMDAR signaling. The triplet puzzle theory also suggests the formation of putative D2R-CCR2, D2R-CXCR4, and D2R-IL1R2 heteromers in mild neuroinflammation in view of their demonstrated sets of Leu-Tyr-Ser (LYS), Leu-Pro-Phe (LPF), and/or Ser-Leu-Ala (SLA) triplet homologies. These D2R heteroreceptor complexes may also contribute to schizophrenia-like symptoms in mild neuroinflammation by enhancing D2R protomer function.
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The introduction of allosteric receptor-receptor interactions in G protein-coupled receptor (GPCR) heteroreceptor complexes of the central nervous system (CNS) gave a new dimension to brain integration and neuropsychopharmacology. The molecular basis of learning and memory was proposed to be based on the reorganization of the homo- and heteroreceptor complexes in the postjunctional membrane of synapses. Long-term memory may be created by the transformation of parts of the heteroreceptor complexes into unique transcription factors which can lead to the formation of specific adapter proteins. The observation of the GPCR heterodimer network (GPCR-HetNet) indicated that the allosteric receptor-receptor interactions dramatically increase GPCR diversity and biased recognition and signaling leading to enhanced specificity in signaling. Dysfunction of the GPCR heteroreceptor complexes can lead to brain disease. The findings of serotonin (5-HT) hetero and isoreceptor complexes in the brain over the last decade give new targets for drug development in major depression. Neuromodulation of neuronal networks in depression via 5-HT, galanin peptides and zinc involve a number of GPCR heteroreceptor complexes in the raphe-hippocampal system: GalR1-5-HT1A, GalR1-5-HT1A-GPR39, GalR1-GalR2, and putative GalR1-GalR2-5-HT1A heteroreceptor complexes. The 5-HT1A receptor protomer remains a receptor enhancing antidepressant actions through its participation in hetero- and homoreceptor complexes listed above in balance with each other. In depression, neuromodulation of neuronal networks in the raphe-hippocampal system and the cortical regions via 5-HT and fibroblast growth factor 2 involves either FGFR1-5-HT1A heteroreceptor complexes or the 5-HT isoreceptor complexes such as 5-HT1A-5-HT7 and 5-HT1A-5-HT2A. Neuromodulation of neuronal networks in cocaine use disorder via dopamine (DA) and adenosine signals involve A2AR-D2R and A2AR-D2R-Sigma1R heteroreceptor complexes in the dorsal and ventral striatum. The excitatory modulation by A2AR agonists of the ventral striato-pallidal GABA anti-reward system via targeting the A2AR-D2R and A2AR-D2R-Sigma1R heteroreceptor complex holds high promise as a new way to treat cocaine use disorders. Neuromodulation of neuronal networks in schizophrenia via DA, adenosine, glutamate, 5-HT and neurotensin peptides and oxytocin, involving A2AR-D2R, D2R-NMDAR, A2AR-D2R-mGluR5, D2R-5-HT2A and D2R-oxytocinR heteroreceptor complexes opens up a new world of D2R protomer targets in the listed heterocomplexes for treatment of positive, negative and cognitive symptoms of schizophrenia.
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The serotonin and neurotrophic factor hypotheses of depression are well known. The discovery of brain fibroblast growth factor receptor 1 (FGFR1)-5 hydroxytryptamine receptor 1A (5-HT1A) heteroreceptor complexes, and their enhancement of neuroplasticity, offers an integration of these hypotheses at the molecular level. They were first described in the hippocampus and later in midbrain 5-HT neurons, where these heterocomplexes are enriched in 5-HT1A autoreceptors. Combined FGF2 and 5-HT1A agonist treatment increased the formation of these heterocomplexes and the facilitatory allosteric receptor-receptor interactions within them led to the enhancement of FGFR1 signaling and was associated with the development of antidepressant effects. We discuss these findings with regard to a theory of motifs critically involved in these interactions and suggest that these complexes represent novel targets for antidepressants.
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Trastorno Depresivo Mayor/metabolismo , Neurociencias/tendencias , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Animales , Trastorno Depresivo Mayor/tratamiento farmacológico , HumanosRESUMEN
Biochemical studies had previously demonstrated examples of heteromerization between opioid and chemokine receptors. Based on the triplet puzzle theory, it has been discovered that opioid receptors are structurally more closely related to chemokine receptors than to other class A G-protein-coupled receptors. Their similarity is established in terms of the number of triplet homologies Asn-Leu-Ala, Thr-Leu-Pro, and Tyr-Ala-Phe in the amino acid code of extensive numbers of members of these two receptor groups. Such widespread similarities probably mean that many opioid and chemokine receptor subtypes utilize some of these mutual triplets to form heteromers. The findings underline that heteromerization among these two receptor groups can represent a major general mechanism for significant interactions between opioid peptides and chemokines in pain and neuroinflammation within the neural-glial networks of the CNS including immune cells.
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Receptores de Quimiocina/metabolismo , Receptores Opioides/metabolismo , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Receptores de Quimiocina/genética , Receptores Opioides/genéticaRESUMEN
New findings show existence of FGFR1-5-HT1A heteroreceptor complexes in 5-HT nerve cells of the dorsal and median raphe nuclei of the rat midbrain and hippocampus. Synergistic receptor-receptor interactions in these receptor complexes indicated their enhancing role in hippocampal plasticity. The existence of FGFR1-5-HT1A heteroreceptor complexes also in midbrain raphe 5-HT nerve cells open up the possibility that antidepressant drugs by increasing extracellular 5-HT levels can cause an activation of the FGF-2/FGFR1 mechanism in these nerve cells as well. Therefore, the agonist modulation of the FGFR1-5-HT1A heteroreceptor complexes and their specific role is now determined in rat medullary raphe RN33B cells and in the caudal midline raphe area of the midbrain rich in 5-HT nerve cells. The combined i.c.v. treatment with FGF-2 and the 5-HT1A agonist 8-OHDPAT synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and in the RN33B cells. Cotreatment with FGF2 and the 5-HT1A agonist induced RN33B cell differentiation as seen from development of an increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TMV but not by TMII of the 5-HT1A receptor. Taken together, the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A heteroreceptor complex in the midbrain raphe 5-HT nerve cells appears to have also a trophic role in the central 5-HT neuron systems besides playing a key role in reducing the firing of these neurons.
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Mesencéfalo/citología , Plasticidad Neuronal , Neuronas/citología , Mapas de Interacción de Proteínas , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , Serotonina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/administración & dosificación , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Línea Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factor 2 de Crecimiento de Fibroblastos/administración & dosificación , Factor 2 de Crecimiento de Fibroblastos/farmacología , Mesencéfalo/efectos de los fármacos , Mesencéfalo/fisiología , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Ratas , Agonistas del Receptor de Serotonina 5-HT1/administración & dosificación , Agonistas del Receptor de Serotonina 5-HT1/farmacologíaRESUMEN
Two major types of intercellular communication are found in the central nervous system (CNS), namely wiring transmission (point-to-point communication, the prototype being synaptic transmission with axons and terminals) and volume transmission (VT; communication in the extracellular fluid and in the cerebrospinal fluid (CSF)) involving large numbers of cells in the CNS. Volume and synaptic transmission become integrated inter alia through the ability of their chemical signals to activate different types of receptor protomers in heteroreceptor complexes located synaptically or extrasynaptically in the plasma membrane. The demonstration of extracellular dopamine (DA) and serotonin (5-HT) fluorescence around the DA and 5-HT nerve cell bodies with the Falck-Hillarp formaldehyde fluorescence method after treatment with amphetamine and chlorimipramine, respectively, gave the first indications of the existence of VT in the brain, at least at the soma level. There exist different forms of VT. Early studies on VT only involved spread including diffusion and flow of soluble biological signals, especially transmitters and modulators, a communication called extrasynaptic (short distance) and long distance (paraaxonal and paravascular and CSF pathways) VT. Also, the extracellular vesicle type of VT was demonstrated. The exosomes (endosome-derived vesicles) appear to be the major vesicular carriers for VT but the larger microvesicles also participate. Both mainly originate at the soma-dendritic level. They can transfer lipids and proteins, including receptors, Rab GTPases, tetraspanins, cholesterol, sphingolipids and ceramide. Within them there are also subsets of mRNAs and non-coding regulatory microRNAs. At the soma-dendritic membrane, sets of dynamic postsynaptic heteroreceptor complexes (built up of different types of physically interacting receptors and proteins) involving inter alia G protein-coupled receptors including autoreceptors, ion channel receptors and receptor tyrosine kinases are hypothesized to be the molecular basis for learning and memory. At nerve terminals, the presynaptic heteroreceptor complexes are postulated to undergo plastic changes to maintain the pattern of multiple transmitter release reflecting the firing pattern to be learned by the heteroreceptor complexes in the postsynaptic membrane.
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Comunicación Celular/fisiología , Membrana Celular/metabolismo , Sistema Nervioso Central/fisiología , Vesículas Extracelulares/fisiología , Red Nerviosa/metabolismo , Neuroglía/metabolismo , Neurotransmisores/metabolismo , Células Receptoras Sensoriales/metabolismo , Difusión , HumanosRESUMEN
The ascending midbrain 5-HT neurons known to contain 5-HT1A autoreceptors may be dysregulated in depression due to a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by co-location of the FGFR1 and 5-HT1A immunoreactivities in midbrain raphe 5-HT cells, evidence for the existence of FGFR1-5-HT1A heteroreceptor complexes were obtained in the dorsal and median raphe nuclei of the Sprague-Dawley rat. Their existence in the rat medullary raphe RN33B cell cultures was also established. After combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA positive clusters was found in the RN33B cells. Similar results were reached upon coactivation by agonists in HEK293T cells using the Fluorescent Resonance Energy Transfer (FRET) technique resulting in increased FRETmax and reduced FRET50 values. The heteroreceptor complex formation was dependent on TMV of the 5-HT1A receptor since it was blocked by incubation with TMV but not with TMII. Taken together, the 5-HT1A autoreceptors by being recruited into a FGFR1-5-HT1A heteroreceptor complex in the midbrain raphe 5-HT nerve cells may develop a novel function, namely a trophic role in many midbrain 5-HT neuron systems originating from the dorsal and medianus raphe nuclei.
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Regulación de la Expresión Génica , Núcleos del Rafe Mesencefálico/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor de Serotonina 5-HT1A/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralin/farmacología , Animales , Dimerización , Factor 2 de Crecimiento de Fibroblastos/farmacología , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Masculino , Neuronas/metabolismo , Péptidos/química , Unión Proteica , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismoRESUMEN
This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their receptor-receptor interactions and relevance for the treatment of schizophrenia. A "guide-and-clasp" manner for receptor-receptor interactions is proposed where "adhesive guides" may be amino acid triplet homologies, which were determined for different kinds of D2 heteroreceptor complexes. The first putative D2 heteroreceptor complex to be discovered in relation to schizophrenia was the A2A-D2 heteroreceptor complex where antagonistic A2A-D2 receptor-receptor interactions were demonstrated after A2A agonist treatment in the ventral striatum. The A2A agonist CGS 21680 with atypical antipsychotic properties may at least in part act by increasing ß-arrestin2 signaling over the D2 protomer in the A2A-D2 heteroreceptor complex in the ventral striatum. The antagonistic NTS1-D2 interactions in the NTS1-D2 heteroreceptor complex in the ventral striatum are proposed as one molecular mechanism for the potential antipsychotic effects of NT. Indications were obtained that the psychotic actions of the 5-HT2AR hallucinogens LSD and DOI can involve enhancement of D2R protomer signaling via a biased agonist action at the 5-HT2A protomer in the D2-5-HT2A heteroreceptor complex in the ventral striatum. Facilitatory allosteric D2likeR-OTR interactions in heteroreceptor complexes in nucleus accumbens may have a role in social and emotional behaviors. By blocking the D2 protomers of these heteroreceptor complexes, antipsychotics can fail to reduce the negative symptoms of schizophrenia. The discovery of different types of D2 heteroreceptor complexes gives an increased understanding of molecular mechanisms involved in causing schizophrenia and new strategies for its treatment and understanding the side effects of antipsychotics.
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Antipsicóticos/farmacología , Receptores de Dopamina D2/metabolismo , Esquizofrenia/metabolismo , Estriado Ventral/metabolismo , Animales , Dopamina/metabolismo , Humanos , Receptores de Dopamina D2/química , Esquizofrenia/tratamiento farmacológicoRESUMEN
There is serious interest in understanding the dynamics of the receptor-receptor and receptor-protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor-receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor-receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology.
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Descubrimiento de Drogas/métodos , Terapia Molecular Dirigida/métodos , Mapeo de Interacción de Proteínas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animales , Antidepresivos/farmacología , Antidepresivos/uso terapéutico , Antiparkinsonianos/farmacología , Antiparkinsonianos/uso terapéutico , Antipsicóticos/farmacología , Antipsicóticos/uso terapéutico , Cocaína/efectos adversos , Cocaína/farmacología , Trastornos Relacionados con Cocaína/tratamiento farmacológico , Trastornos Relacionados con Cocaína/metabolismo , Depresión/tratamiento farmacológico , Depresión/metabolismo , Humanos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Esquizofrenia/tratamiento farmacológico , Esquizofrenia/metabolismoRESUMEN
The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR) exerts an antagonistic allosteric influence on the mu opioid receptor (MOR) function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via ß -arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR) heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor)-TRPV1 (capsaicin receptor) heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia.
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Based on our theory, main triplets of amino acid residues have been discovered in cell-adhesion receptors (integrins) of marine sponges, which participate as homologies in the interface between two major immune molecules, MHC class I (MHCI) and CD8αß. They appear as homologies also in several human neural receptor heteromers and subunits. The obtained results probably mean that neural and immune receptors also utilize these structural integrin triplets to form heteromers and ion channels, which are required for a tuned and integrated intracellular and intercellular communication and a communication between cells and the extracellular matrix with an origin in sponges, the oldest multicellular animals.
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Biochemical, histochemical and coimmunoprecipitation experiments have indicated the existence of antagonistic dopamine D2 (D2R) and neurotensin 1 (NTS1R) receptor-receptor interactions in the dorsal and ventral striatum indicating a potential role of these receptor-receptor interactions in Parkinson's disease and schizophrenia. By means of Bioluminiscence Resonance energy transfer (BRET(2)) evidence has for the first time been obtained in the current study for the existence of both D2LR/NTS1R and D2SR/NTS1R heteromers in living HEK293T cells. Through confocal laser microscopy the NTS1R(GFP2) and D2R(YFP) were also shown to be colocated in the plasma membrane of these cells. A bioinformatic analysis suggests the existence of a basic set of three homology protriplets (TVM, DLL and/or LRA) in the two participating receptors which may contribute to the formation of the D2R/NTS1R heteromers by participating in guide-clasp interactions in the receptor interface. The CREB reporter gene assay indicated that the neurotensin receptor agonist JMV 449 markedly reduced the potency of the D2R like agonist quinpirole to inhibit the forskolin induced increase of the CREB signal. In contrast, the neurotensin agonist was found to markedly increase the quinpirole potency to activate the MAPK pathway as also studied with luciferase reporter gene assay measuring the degree of SRE activity as well as with ERK1/2 phosphorylation assays. These dynamic changes in D2R signaling produced by the neurotensin receptor agonist may involve antagonistic allosteric receptor-receptor interactions in the D2LR-NTS1R heteromers at the plasma membrane level (CREB pathway) and synergistic interactions in PKC activation at the cytoplasmatic level (MAPK pathway).
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Multimerización de Proteína , Receptores de Dopamina D2/química , Receptores de Neurotensina/química , Transducción de Señal , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Membrana Celular/metabolismo , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Modelos Moleculares , Oligopéptidos/farmacología , Fosforilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Pirazoles/farmacología , Quinolinas/farmacología , Quinpirol/farmacología , Racloprida/farmacología , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) play critical roles in cellular processes and signaling and have been shown to form heteromers with diverge biochemical and/or pharmacological activities that are different from those of the corresponding monomers or homomers. However, despite extensive experimental results supporting the formation of GPCR heteromers in heterologous systems, the existence of such receptor heterocomplexes in the brain remains largely unknown, mostly because of the lack of appropriate methodology. Herein, we describe the in situ proximity ligation assay procedure underlining its high selectivity and sensitivity to image GPCR heteromers with confocal microscopy in brain sections. We describe here how the assay is performed and discuss advantages and disadvantages of this method compared with other available techniques.
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Encéfalo/metabolismo , Receptores Acoplados a Proteínas G/análisis , Receptores Acoplados a Proteínas G/metabolismo , Regulación Alostérica , Animales , Encéfalo/ultraestructura , Humanos , Microscopía Confocal/métodos , Modelos Moleculares , Plasticidad Neuronal , Mapeo de Interacción de Proteínas/métodos , Multimerización de Proteína , Receptores de Factores de Crecimiento de Fibroblastos/análisis , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Serotonina/análisis , Receptores de Serotonina/metabolismo , Transducción de SeñalRESUMEN
Galanin receptor (GalR) subtypes 1-3 linked to central galanin neurons may form heteromers with each other and other types of G protein-coupled receptors in the central nervous system (CNS). These heteromers may be one molecular mechanism for galanin peptides and their N-terminal fragments (gal 1-15) to modulate the function of different types of glia-neuronal networks in the CNS, especially the emotional and the cardiovascular networks. GalR-5-HT1A heteromers likely exist with antagonistic GalR-5-HT1A receptor-receptor interactions in the ascending midbrain raphe 5-HT neuron systems and their target regions. They represent a novel target for antidepressant drugs. Evidence is given for the existence of GalR1-5-HT1A heteromers in cellular models with trans-inhibition of the protomer signaling. A GalR1-GalR2 heteromer is proposed to be a galanin N-terminal fragment preferring receptor (1-15) in the CNS. Furthermore, a GalR1-GalR2-5-HT1A heterotrimer is postulated to explain why only galanin (1-15) but not galanin (1-29) can antagonistically modulate the 5-HT1A receptors in the dorsal hippocampus rich in gal fragment binding sites. The results underline a putative role of different types of GalR-5-HT1A heteroreceptor complexes in depression. GalR antagonists may also have therapeutic actions in depression by blocking the antagonistic GalR-NPYY1 receptor interactions in putative GalR-NPYY1 receptor heteromers in the CNS resulting in increases in NPYY1 transmission and antidepressant effects. In contrast the galanin fragment receptor (a postulated GalR1-GalR2 heteromer) appears to be linked to the NPYY2 receptor enhancing the affinity of the NPYY2 binding sites in a putative GalR1-GalR2-NPYY2 heterotrimer. Finally, putative GalR-α2-adrenoreceptor heteromers with antagonistic receptor-receptor interactions may be a widespread mechanism in the CNS for integration of galanin and noradrenaline signals also of likely relevance for depression.
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Extrasynaptic neurotransmission is an important short distance form of volume transmission (VT) and describes the extracellular diffusion of transmitters and modulators after synaptic spillover or extrasynaptic release in the local circuit regions binding to and activating mainly extrasynaptic neuronal and glial receptors in the neuroglial networks of the brain. Receptor-receptor interactions in G protein-coupled receptor (GPCR) heteromers play a major role, on dendritic spines and nerve terminals including glutamate synapses, in the integrative processes of the extrasynaptic signaling. Heteromeric complexes between GPCR and ion-channel receptors play a special role in the integration of the synaptic and extrasynaptic signals. Changes in extracellular concentrations of the classical synaptic neurotransmitters glutamate and GABA found with microdialysis is likely an expression of the activity of the neuron-astrocyte unit of the brain and can be used as an index of VT-mediated actions of these two neurotransmitters in the brain. Thus, the activity of neurons may be functionally linked to the activity of astrocytes, which may release glutamate and GABA to the extracellular space where extrasynaptic glutamate and GABA receptors do exist. Wiring transmission (WT) and VT are fundamental properties of all neurons of the CNS but the balance between WT and VT varies from one nerve cell population to the other. The focus is on the striatal cellular networks, and the WT and VT and their integration via receptor heteromers are described in the GABA projection neurons, the glutamate, dopamine, 5-hydroxytryptamine (5-HT) and histamine striatal afferents, the cholinergic interneurons, and different types of GABA interneurons. In addition, the role in these networks of VT signaling of the energy-dependent modulator adenosine and of endocannabinoids mainly formed in the striatal projection neurons will be underlined to understand the communication in the striatal cellular networks.
RESUMEN
The evidence for the existence of receptor heteromers opens up a new field for a better understanding of neural transmission. Based on our theory, we have discovered main triplets of amino acid residues in cell-adhesion receptors of marine sponges, which appear also as homologies in several dopamine D2 receptor heteromers of human brain. The obtained results probably mean a general molecular mechanism for receptor-receptor interactions in heteromers originated from the lowest animals (marine sponges).
Asunto(s)
Organismos Acuáticos/metabolismo , Integrinas/química , Poríferos/metabolismo , Multimerización de Proteína , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Evolución Molecular , Humanos , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Based on our theory, we have discovered main triplets of amino acid residues in cell-adhesion receptors of marine sponges, which appear also as homologies in several receptor heteromers of human brain. The obtained results strengthen our hypothesis that these triplets may "guide-and-clasp" receptor-receptor interactions.
Asunto(s)
Encéfalo/fisiología , Evolución Molecular , Geodia/genética , Integrinas/genética , Receptores de Superficie Celular/genética , Animales , Humanos , Datos de Secuencia Molecular , Filogenia , Receptor de Colecistoquinina B/genética , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Receptor del Glutamato Metabotropico 5 , Receptor de Serotonina 5-HT1A/genética , Receptor de Serotonina 5-HT1B/genética , Receptor de Serotonina 5-HT1D/genética , Receptores Adrenérgicos alfa 2/genética , Receptores de GABA-B/genética , Receptores de Glutamato Metabotrópico/genética , Homología de Secuencia de AminoácidoRESUMEN
The ascending midbrain 5-HT neurons to the forebrain may be dysregulated in depression and have a reduced trophic support. With in situ proximity ligation assay (PLA) and supported by coimmunoprecipitation and colocation of the FGFR1 and 5-HT1A immunoreactivities in the midbrain raphe cells, evidence for the existence of FGFR1-5-HT1A receptor heterocomplexes in the dorsal and median raphe nuclei of the Sprague Dawley rat as well as in the rat medullary raphe RN33B cells has been obtained. Especially after combined FGF-2 and 8-OH-DPAT treatment, a marked and significant increase in PLA clusters was found in the RN33B cells. Similar results were reached with the FRET technique in HEK293T cells, where TM-V of the 5HT1A receptor was found to be part of the receptor interface. The combined treatment with FGF-2 and the 5-HT1A agonist also synergistically increased FGFR1 and ERK1/2 phosphorylation in the raphe midline area of the midbrain and the RN33B cells as well as their differentiation, as seen from development of the increased number and length of extensions per cell and their increased 5-HT immunoreactivity. These signaling and differentiation events were dependent on the receptor interface since they were blocked by incubation with TM-V but not by TM-II. Together, the results indicate that the 5-HT1A autoreceptors by being part of a FGFR1-5-HT1A receptor heterocomplex in the midbrain raphe 5-HT nerve cells appear to have a trophic role in the central 5-HT neuron systems in addition to playing a key role in reducing the firing of these neurons.